Complement is activated by three pathways, the classical pathway, the alternative pathway, and the recently discovered lectin pathway, all of which lead to the formation of the cytolytic membrane attack complex, C5b-9. Following complement activation, the biologically active peptides C5a and C3a elicit a number of proinflammatory effects, such as chemotaxis of leukocytes, degranulation of phagocytic cells, mast cells, and basophils, smooth muscle contraction, and increase of vascular permeability. Upon activation by these complement products; the inflammatory response is further amplified by subsequent generation of toxic oxygen radicals, induction of synthesis, and release of arachidonic acid metabolites and cytokines. Consequently, an (over)activated complement system presents a considerable risk of harming the host by directly and indirectly mediating inflammatory tissue destruction. The key proteins involved in the activation of the alternative pathway are factors P, B and factor D. These proteins work together to amplify the activation of C3, which then leads to the initiation of a number of inflammatory events.
The trigger of the alternative complement pathway is artificial surfaces including bacteria, parasites, viruses or fungi. Alternative pathway activation is triggered when factor B binds to C3b or C3H2O. This complex is then cleaved by factor D to produce C3 convertase (C3bBb). The alternative pathway C3 convertase is stabilized by the binding of properdin, extending its half-life six-to ten-fold. An amplification loop is established as the C3 convertase generates increasing amounts of C3b. The classical pathway can also generate C3b, which can engage the alternative pathway by binding factor B. Addition of C3b to the C3 convertase (PC3bBb) leads to the formation of the alternative pathway C5 convertase, PC3bBbC3b. All three pathways, the classical, lectin, and alternative converge at C3, which is cleaved by C3 convertase to form C3a with multiple pro-inflammatory effects. C5a and C5b are formed by the cleavage of C5. The C5b associates with the cell membrane and ultimately becomes the part of membrane attack complex C5b-9 (MAC), which is now thought to play an important role in inflammation as well as a lytic pore-forming complex.
Split products of both C3 and C5 designated as C3a and C5a are potent anaphylatoxins and are responsible for activating neutrophils, monocytes, and platelets. These activated cells indiscriminately release destructive enzymes that have the capacity to cause significant organ damage. Anaphylatoxins can significantly amplify inflammatory responses by inducing the release of numerous additional inflammatory mediators, including cytokines, hydrolytic enzymes, arachidonic acid metabolites, and reactive oxygen species from neutrophils and monocytes. C3a is a potent anaphylatoxin which can initiate detrimental events, including the release of pro-inflammatory cytokines (Takabayashi, T., et al., A new biologic role for C3a and C3a desArg: regulation of TNF-alpha and IL-1 beta synthesis. J Immunol, 1996. 156(9): p. 3455-60) and prostaglandins (Howard, R. J., et al., Effects of cardiopulmonary bypass on pulmonary leukostasis and complement activation. Arch Surg, 1988. 123(12): p. 1496-501) from monocytes, activation of monocytes (Haeffner-Cavaillon, N., et al., C3a(C3adesArg) induces production and release of interleukin 1 by cultured human monocytes. J Immunol, 1987. 139(3): p. 794-9; Rinder, C. S., et al., Role of C3 cleavage in monocyte activation during extracorporeal circulation. Circulation, 1999. 100(5): p. 553-8) histamine release from mast cells, and de-granulation of eosinophils (Krug, N., et al., Complement factors C3a and C5a are increased in bronchoalveolar lavage fluid after segmental allergen provocation in subjects with asthma. Am J Respir Crit Care Med, 2001. 164(10 Pt 1): p. 1841-3). C3a plays a significant role in cognition and memory (Hugli, T. E., The structural basis for anaphylatoxin and chemotactic functions of C3a, C4a, and C5a. Crit Rev Immunol, 1981. 1(4): p. 321-66; van Beek, J., K. Elward, and P. Gasque, Activation of complement in the central nervous system: roles in neurodegeneration and neuroprotection. Ann N Y Acad Sci, 2003. 992: p. 56-71). C3a receptors are mainly found on neutrophils and monocytes (Gerardy-Schahn, R., et al., Characterization of C3a receptor-proteins on guinea pig platelets and human polymorphonuclear leukocytes. European Journal of Immunology, 1989. 19(6): p. 1095; Cecic, I., J. Sun, and M. Korbelik, Role of complement anaphylatoxin C3a in photodynamic therapy-elicited engagement of host neutrophils and other immune cells. Photochem Photobiol, 2006. 82(2): p. 558-62). Increased levels of C3a in the circulation have been found in diseases such as the adult respiratory distress syndrome (Solomkin, J. S., et al., Complement activation and clearance in acute illness and injury: evidence for C5a as a cell-directed mediator of the adult respiratory distress syndrome in man. Surgery, 1985. 97(6): p. 668-78; Abe, M., [Complement activation and inflammation]. Rinsho Byori, 2006. 54(7): p. 744-56), rheumatoid arthritis (Rinsho Byori, 2006. 54(7): p. 744-56), psoriasis (Rinsho Byori, 2006. 54(7): p. 744-56; Kapp, A., H. Wokalek, and E. Schopf, Involvement of complement in psoriasis and atopic dermatitis—measurement of C3a and C5a, C3, C4 and C1 inactivator. Arch Dermatol Res, 1985. 277(5): p. 359-61) and atopic dermatitis (Arch Dermatol Res, 1985. 277(5): p. 359-61). C3a is also found intralesionally in inflammatory diseases, e.g. psoriasis (Takematsu, H., K. Ohkohchi, and H. Tagami, Demonstration of anaphylatoxins C3a, C4a and C5a in the scales of psoriasis and inflammatory pustular dermatoses. Br J Dermatol, 1986. 114(1): p. 1-6), eczema (Br J Dermatol, 1986. 114(1): p. 1-6), and asthma (Am J Respir Crit Care Med, 2001. 164(10 Pt 1): p. 1841-3; Rinsho Byori, 2006. 54(7): p. 744-56; Marc, M. M., et al., Complement factors c3a, c4a, and c5a in chronic obstructive pulmonary disease and asthma. American Journal of Respiratory Cell and Molecular Biology, 2004. 31(2): p. 216; Zaidi, A. K., et al., Response to C3a, mast cells, and asthma. Faseb J, 2006. 20(2): p. 199; Humbles, A. A., et al., A role for the C3a anaphylatoxin receptor in the effector phase of asthma. Nature, 2000. 406(6799): p. 998-1001).
Inappropriate activation of the complement system contributes to pathogenesis of numerous acute and chronic disease states, including Myocardial Infarction, Reperfusion Injury, Stroke, ARDS, Hemodialysis, Plasmapheresis, leukopheresis, Cardiopulmonary bypass, Rheumatoid arthritis, systemic lupus erythematosus (SLE), osteoarthritis, lupus, membranous nephritis, Myasthenia Gravis, pancreatitis, Septic Shock, Multiple Sclerosis, Alzheimer's Disease, Traumatic Brain Injury, Spinal Cord Injury, Neuropathic Pain, Neurological Injury, spontaneous abortion, miscarriages, Transplant Rejection, Asthma, Cancer, Thermal Burn. Despite the role of complement in several disease indications, no drug currently exists to downregulate this complement activation.
The following are disorders associated with complement activation: systemic inflammatory reaction syndrome, multiple organ dysfunction syndrome, ischemia-reperfusion syndrome, angioedema, capillary leak syndrome, hyperacute and acute graft rejection, vasculitis, nephritis, autoimmune disorders (e.g., SLE, rheumatoid arthritis, and myasthenia gravis), biomaterial incompatibility (e.g., following dialysis or cardiopulmonary bypass), and severe trauma, burn, and sepsis. Only recently has complement also been implicated in neurodegenerative disorders, such as Alzheimer's disease (Ann N Y Acad Sci, 2003. 992: p. 56-71; Zanjani, H., et al., Complement activation in very early Alzheimer disease. Alzheimer Disease and Associated Disorders, 2005. 19(2): p. 55), multiple sclerosis (Sanders, M. E., et al., Activated terminal complement in cerebrospinal fluid in Guillain-Barre syndrome and multiple sclerosis. J Immunol, 1986. 136(12): p. 4456-9), Guillain-Barré syndrome (J Immunol, 1986. 136(12): p. 4456-9), traumatic brain injury and spinal cord injury (Bellander, B. M., et al., Complement activation in the human brain after traumatic head injury. J Neurotrauma, 2001. 18(12): p. 1295-311; Leinhase, I., et al., Inhibition of the alternative complement activation pathway in traumatic brain injury by a monoclonal anti-factor B antibody: a randomized placebo-controlled study in mice. J Neuroinflammation, 2007. 4: p. 13; Stahel, P. F., et al., Intrathecal levels of complement-derived soluble membrane attack complex (sC5b-9) correlate with blood-brain barrier dysfunction in patients with traumatic brain injury. J Neurotrauma, 2001. 18(8): p. 773-81; Schmidt, O. I., et al., [The relevance of the inflammatory response in the injured brain]. Orthopade, 2007. 36(3): p. 248, 250-8; Morganti-Kossmann, M. C., et al., Role of cerebral inflammation after traumatic brain injury: a revisited concept. Shock, 2001. 16(3): p. 165-77; Stahel, P. F., M. C. Morganti-Kossmann, and T. Kossmann, The role of the complement system in traumatic brain injury. Brain Res Brain Res Rev, 1998. 27(3): p. 243-56). Furthermore, activation of complement is a critical event in the pathogenesis of sepsis and septic shock (Bengston, A., Heideman, M, Anaphylatoxin formation in sepsis. Arch Surg, 1988. 123: p. 645-649; Laudes, I. J., et al., Anti-c5a ameliorates coagulation/fibrinolytic protein changes in a rat model of sepsis. American Journal Of Pathology, 2002. 160(5): p. 1867; Hack, C. E., et al., A model for the interplay of inflammatory mediators in sepsis—a study in 48 patients. Intensive Care Med, 1990. 16 Suppl 3: p. S187-91). Complement activation after polytrauma substantially contributes to the development of systemic inflammatory reaction syndrome and multiple organ failure (Kirschfink, M., Controlling the complement system in inflammation. Immunopharmacology, 1997. 38(1-2): p. 51-62; Sistino, J. J. and J. R. Acsell, Systemic inflammatory response syndrome (SIRS) following emergency cardiopulmonary bypass: a case report and literature review. J Extra Corpor Technol, 1999. 31(1): p. 37-43). In recent years, complement has been recognized as a major effector mechanism of reperfusion injury. The inflammatory response induced by artificial surfaces in hemodialysis and extracorporeal circuits may lead to organ dysfunction. Here, complement activation has been shown to be associated with transient neutropenia, pulmonary vascular leukostasis, and occasionally, anaphylactic shock of variable severity in patients undergoing hemodialysis or cardiopulmonary bypass (Immunopharmacology, 1997. 38(1-2): p. 51-62; Chenoweth, D. E., Anaphylatoxin formation in extracorporeal circuits. Complement, 1986. 3(3): p. 152-65; Hammerschmidt, D. E., et al., Complement activation and neutropenia occurring during cardiopulmonary bypass. J Thorac Cardiovasc Surg, 1981. 81(3): p. 370-7; Lin, Y. F., et al., Cytokine production during hemodialysis: effects of dialytic membrane and complement activation. Am J Nephrol, 1996. 16(4): p. 293-9; Chenoweth, D. E., et al., Complement activation during cardiopulmonary bypass: evidence for generation of C3a and C5a anaphylatoxins. N Engl J Med, 1981. 304(9): p. 497-503; Tamiya, T., et al., Complement activation in cardiopulmonary bypass, with special reference to anaphylatoxin production in membrane and bubble oxygenators. Ann Thorac Surg, 1988. 46(1): p. 47-57; Chello, M., et al., Complement and neutrophil activation during cardiopulmonary bypass: a randomized comparison of hypothermic and normothermic circulation. Eur J Cardiothorac Surg, 1997. 11(1): p. 162-8; Levy, J. H. and K. A. Tanaka, Inflammatory response to cardiopulmonary bypass. Ann Thorac Surg, 2003. 75(2): p. S715-20).
In recent years, great progress has been made in complement analysis to better define disease severity, evolution, and response to therapy. Modern diagnostic technologies, which focus on the quantification of complement-derived split products or protein-protein complexes, now provide comprehensive insight into the activation state of the system. In certain vasculitides and kidney diseases, a substantial activation and consumption of C3 due to defective alternative pathway regulation can be observed. Patients suffering from MPGN, show low levels of CH50, AH50, and C3. This results from a continuous C3 activation due to an autoantibody, termed C3 nephritic factor (C3NeF), which stabilizes the labile C3bBb complex (Jelezarova, E., et al., A C3 convertase assay for nephritic factor functional activity. J Immunol Methods, 2001. 251(1-2): p. 45-52).
Role of Coagulation and Fibrinolysis in CPB
Coagulation/and platelet activation cascades are initiated during cardiopulmonary bypass (CPB) based on the reported production of thrombin and platelet activation markers upon blood exposure to artificial surfaces (Edmunds, L. H., Jr. and R. W. Colman, Thrombin during cardiopulmonary bypass. Ann Thorac Surg, 2006. 82(6): p. 2315-22). Thrombotic and bleeding complications seem to be caused by generation of thrombin during cardiopulmonary bypass. Thrombin generation and the fibrinolytic response primarily involve the extrinsic and intrinsic coagulation pathways, the contact and fibrinolytic plasma protein systems, and platelets, monocytes, and endothelial cells. Thrombin is generated because of extracorporeal circulation and varies with the amount and type of anticoagulant used.
Due to increasing evidence, there are indications that the wound is the major generation source of thrombin during CPB and clinical cardiac surgery. Endothelial cells are activated by circulating thrombin, to produce tissue plasminogen activator (t-PA), which binds fibrin. Endothelial cells are the principal source of t-PA, which when combined with fibrin and plasminogen, cleaves plasminogen to plasmin; plasmin cleaves fibrin. This reaction produces the useful marker of fibrinolysis, protein fragment, and D-dimer. D-dimer increases during extracorporeal perfusion, indicating ongoing thrombin production, fibrin formation, and fibrinolysis.
Heparin, the anticoagulant drug most often employed first in the prevention and treatment of thromboembolic diseases, causes heparin-induced thrombocytopenia (HIT) a special class of platelet thrombosis that occurs as an immune response to the drug. The morbidity and mortality of HIT patients remains high, while standard treatment of HIT involves discontinuing heparin and the use of an alternative anticoagulant, such as a thrombin inhibitor, followed by close platelet count monitoring for the recovery. Recently, despite the use of alternatives, a standard dose of GPIIb/IIIa antagonist, combined with a lowered dose of thrombin inhibitor to minimize hemorrhagic events, was used to treat HIT thrombosis (Walenga, J. M., et al., Clinical experience with combined treatment of thrombin inhibitors and GPIIb/IIIa inhibitors in patients with HIT. Semin Thromb Hemost, 1999. 25 Suppl 1: p. 77-81). A fibrinolytic-resistant re-thrombosis that is platelet-rich often occurs, despite tendencies of initial thrombosis of the coronary arteries being susceptible to treatment with fibrin-dissolving agents, such as tissue plasminogen activator or streptokinase. Combination of thrombin inhibitors with the use of fast-acting antiplatelet drugs such as GPIIb/IIIa antagonists is often necessary to control the local generation of active thrombin. However, combination of improved anticoagulants with the GPIIb/IIIa antagonists are needed in the treatment of HIT and other thrombo-embolic disorders. Surface-bound thrombin at residual levels amplifies the generation of systemic thrombin by catalyzing prothrombin consumption via the thrombin feedback loop at the site of vascular injury (Fenton, J. W., 2nd, et al., Understanding thrombin and hemostasis. Hematol Oncol Clin North Am, 1993. 7(6): p. 1107-19; Ofosu, F. A., Anticoagulant actions of tissue factor pathway inhibitor on tissue-factor-dependent plasma coagulation. Semin Thromb Hemost, 1995. 21(2): p. 240-4; Ofosu, F. A., et al., Inhibition of the amplification reactions of blood coagulation by site-specific inhibitors of alpha-thrombin. Biochem J, 1992. 283(Pt 3): p. 893-7). Moreover, when thrombin is generated in response to an injury or disease, it can be found in the systemic circulation or fluid phase, as well as associated with the fibrin clot, cell surfaces, such as platelets, the vessel wall, and the biomaterial surfaces of biometric circuits and devices.
Expression of GPIIb/IIIa receptors on the surface of activated platelets greatly enhances their, aggregation and adherence to the fibrin clot and injured vessel wall. Thus, thrombin-activated platelets promote thrombus growth indicating a need for improved thrombin inhibitors with antiplatelet therapies (Eisenberg, P. R. and G. Ghigliotti, Platelet-dependent and procoagulant mechanisms in arterial thrombosis. Int J Cardiol, 1999. 68 Suppl 1: p. S3-10).